Differential gene expression of 36-kDa microfibril-associated glycoprotein (MAGP-36/MFAP4) in rat organs

By using quantitative Western blot analysis and the real time polymerase chain reaction technique, we investigated the differential gene expression of microfibril-associated glycoprotein (MAGP-36) in rat organs. The gene was expressed highly in sites rich in elastic fibers, such as aorta, skin, and esophagus. However, MAGP-36 was also expressed highly in some other sites containing no elastic fibers. In lung and trachea, the expression levels of MAGP-36 mRNA were about seven times higher than those in other elastic tissues, although the protein abundances were almost at the same levels as other elastic tissues. MAGP-36 seemed to be secreted outside these organs. In brain, kidney, and spleen, although the expression levels of MAGP-36 mRNA were low, substantial amounts of MAGP-36 protein were detected. An immunohistochemical study revealed that MAGP-36 was present at the brush border of the S3 segment of proximal tubules in kidney. Since MAGP-36 is known to bind to mannan, MAGP-36 might be involved in mannose transport in the S3 segment. Thus, MAGP-36 might be multifunctional and present in a wide variety of sites in various organs.     

Ultrastructural distribution of 36-kD microfibril-associated glycoprotein (MAGP-36) in human and bovine tissues.

We observed the ultrastructural distribution of MAGP-36 by immunoelectron microscopy in human and bovine tissues. MAGP-36 was present in microfibrils associated with tropoelastin in skin, aorta, and spleen. It was not detected in microfibrils from the ocular zonule and kidney mesangium that were not associated with tropoelastin. In skin, MAGP-36 was present in both early immature elastic fibers and mature elastic fibers. In mature elastic fibers, MAGP-36 was localized around amorphous elastic cores at the elastin-microfibril interface and in electron-dense bundles. Localization of MAGP-36 in elastic fibers coincided with the distribution of lysyl oxidase, an enzyme that plays a pivotal role in the deposition of tropoelastin. These findings suggest that MAGP-36 may be involved in elastogenesis.     

Crosslinked elastic fibers are necessary for low energy loss in the ascending aorta

In the large arteries, it is believed that elastin provides the resistance to stretch at low pressure, while collagen provides the resistance to stretch at high pressure. It is also thought that elastin is responsible for the low energy loss observed with cyclic loading. These tenets are supported through experiments that alter component amounts through protease digestion, vessel remodeling, normal growth, or in different artery types. Genetic engineering provides the opportunity to revisit these tenets through the loss of expression of specific wall components. We used newborn mice lacking elastin (Eln^−/−) or two key proteins (lysyl oxidase, Lox^−/−, or fibulin-4, Fbln4^−/−) that are necessary for the assembly of mechanically-functional elastic fibers to investigate the contributions of elastic fibers to large artery mechanics. We determined component content and organization and quantified the nonlinear and viscoelastic mechanical behavior of Eln^−/−, Lox^−/−, and Fbln4^−/− ascending aorta and their respective controls. We confirmed that the lack of elastin, fibulin-4, or lysyl oxidase leads to absent or highly fragmented elastic fibers in the aortic wall and a 56–97% decrease in crosslinked elastin amounts. We found that the resistance to stretch at low pressure is decreased only in Eln^−/− aorta, confirming the role of elastin in the nonlinear mechanical behavior of the aortic wall. Dissipated energy with cyclic loading and unloading is increased 53–387% in Eln^−/−, Lox^−/−, and Fbln4^−/− aorta, indicating that not only elastin, but properly assembled and crosslinked elastic fibers, are necessary for low energy loss in the aorta.     

Elastic fibers and biomechanics of the aorta: Insights from mouse studies

Elastic fibers are major components of the extracellular matrix (ECM) in the aorta and support a life-long cycling of stretch and recoil. Elastic fibers are formed from mid-gestation throughout early postnatal development and the synthesis is regulated at multiple steps, including coacervation, deposition, cross-linking, and assembly of insoluble elastin onto microfibril scaffolds. To date, more than 30 molecules have been shown to associate with elastic fibers and some of them play a critical role in the formation and maintenance of elastic fibers in vivo. Because the aorta is subjected to high pressure from the left ventricle, elasticity of the aorta provides the Windkessel effect and maintains stable blood flow to distal organs throughout the cardiac cycle. Disruption of elastic fibers due to congenital defects, inflammation, or aging dramatically reduces aortic elasticity and affects overall vessel mechanics. Another important component in the aorta is the vascular smooth muscle cells (SMCs). Elastic fibers and SMCs alternate to create a highly organized medial layer within the aortic wall. The physical connections between elastic fibers and SMCs form the elastin-contractile units and maintain cytoskeletal organization and proper responses of SMCs to mechanical strain. In this review, we revisit the components of elastic fibers and their roles in elastogenesis and how a loss of each component affects biomechanics of the aorta. Finally, we discuss the significance of elastin-contractile units in the maintenance of SMC function based on knowledge obtained from mouse models of human disease.     

Effects of physical exercise on the elasticity and elastic components of the rat aorta

To evaluate the effects of exercise on aortic wall elasticity and elastic components, young male rats underwent various exercise regimes for 16 weeks. In the exercised rats, the aortic incremental elastic modulus decreased significantly when under physiological strain. The aortic content of elastin increased significantly and the calcium content of elastin decreased significantly in the exercised group. The accumulated data from the exercised and sedentary groups revealed that the elastin calcium content was related positively to the incremental elastic modulus. We concluded that physical exercise from an early age decreases the calcium deposit in aortic wall elastin and that this effect probably produced in the exercised rats a distensible aorta.     

Microfibril-associated MAGP-2 stimulates elastic fiber assembly

Elastic fibers are complex structures composed of a tropoelastin inner core and microfibril outer mantle guiding tropoelastin deposition. Microfibrillar proteins mainly include fibrillins and microfibril-associated glycoproteins (MAGPs). MAGP-2 exhibits developmental expression peaking at elastic fiber onset, suggesting that MAGP-2 mediates elastic fiber assembly. To determine whether MAGP-2 regulates elastic fiber assembly, we used an in vitro model featuring doxycycline-regulated cells conditionally overexpressing exogenous MAGP-2 and constitutively expressing enhanced green fluorescent protein-tagged tropoelastin. Analysis by immunofluorescent staining showed that MAGP-2 overexpression dramatically increased elastic fibers levels, independently of extracellular levels of soluble tropoelastin, indicating that MAGP-2 stimulates elastic fiber assembly. This was associated with increased levels of matrix-associated MAGP-2. Electron microscopy showed that MAGP-2 specifically associates with microfibrils and that elastin globules primarily colocalize with MAGP-2-associated microfibrils, suggesting that microfibril-associated MAGP-2 facilitates elastic fiber assembly. MAGP-2 overexpression did not change levels of matrix-associated fibrillin-1, MAGP-1, fibulin-2, fibulin-5, or emilin-1, suggesting that microfibrils and other elastic fiber-associated proteins known to regulate elastogenesis do not mediate MAGP-2-induced elastic fiber assembly. Moreover, mutation analysis showed that MAGP-2 does not stimulate elastic fiber assembly through its RGD motif, suggesting that integrin receptor binding does not mediate MAGP-2-induced elastic fiber assembly. Because MAGP-2 interacts with Jagged-1 that controls cell-matrix interaction and cell motility, two key factors in elastic fiber macroassembly, microfibril-associated MAGP-2 may stimulate elastic fiber macroassembly by targeting the release of elastin globules from the cell membrane onto developing elastic fibers.     

Stability of elastin in the developing mouse aorta: a quantitative radioautographic study

Elastic lamina growth during development and the ultimate stability of elastin in the mouse aortic media was investigated by light and electron microscopic radioautography. Following a single subcutaneous injection of l-[3,4-³H]valine at 3 days of age, animals were killed at 9 subsequent time intervals up to 4 months of age. One day after injection, radioautographic silver grains were primarily observed over the elastic laminae; however, silver grains were also seen over the smooth muscle cells and extracellular matrix. By 21 to 28 days of age, the silver grains were almost exclusively located over the elastic laminae. From 28 days to 4 months of age, the distribution of silver grains appeared relatively unchanged. Quantitation of silver grain number/m² of elastin showed a steady decrease in the concentration of silver grains associated with the elastic laminae from 4 to 21 days of age. After this time, no significant difference in silver grain concentration was observed. Since the initial decrease in grains/m² of elastin corresponds to a period of rapid post-natal growth, the decrease is likely to be a result of dilution of the radiolabel due to new elastin synthesis. With the assumption that little or no significant turnover occurs during this time, a constant growth rate of 4.3% per day was predicted by linear regression analysis. Since no significant difference in the concentration of silver grains was observed from 28 to 118 days of age, no new growth or turnover of elastin can be said to occur during this time period. This is supported by the observation that animals injected with radiolabeled valine at 28 days and 8 months of age showed no significant incorporation of radiolabel into the elastic laminae. The results from this study present the first long-term radioautographic evidence of the stability of aortic elastin and emphasize that initial deposition of elastin and proper assembly of elastic laminae is a critical event in vessel development.     

Microfibrils, elastic anchoring components of the extracellular matrix, are associated with fibronectin in the zonule of Zinn and aorta

Microfibrils are striated tubules that play a role in the formation of elastin fibers by providing a scaffold upon which newly synthesized elastin is deposited. Ultrastructural and staining studies also demonstrate microfibrils that terminate where elastin is sparse or absent in basal laminae, plasma membranes, and the collagenous matrix. The most striking accumulation of microfibrils is found in the zonule of Zinn, the transparent and elastic suspensory ligament of the lens, which contains no elastin. Application of immunocytochemical staining with a peroxidase-antiperoxidase (PAP) procedure demonstrates that fibronectin is associated with the microfibrils of the zonule and aorta. Aggregates of microfibrils are identical to oxytalan ('acid enduring') fibers that have been described in peridontal membranes and other sites subject to mechanical stress and they can be found in sites as disparate as the rabbit zonule, rat hepatic stroma and human cardiac papillary muscle, indicating that microfibrils are a widely distributed connective tissue element with a function that extends beyond elastogenesis; their association with fibronectin and localization suggests that they serve as an elastic anchoring component of the extracellular matrix.     

Multiphoton microscopy observations of 3D elastin and collagen fiber microstructure changes during pressurization in aortic media

Elastin and collagen fibers play important roles in the mechanical properties of aortic media. Because knowledge of local fiber structures is required for detailed analysis of blood vessel wall mechanics, we investigated 3D microstructures of elastin and collagen fibers in thoracic aortas and monitored changes during pressurization. Using multiphoton microscopy, autofluorescence images from elastin and second harmonic generation signals from collagen were acquired in media from rabbit thoracic aortas that were stretched biaxially to restore physiological dimensions. Both elastin and collagen fibers were observed in all longitudinal–circumferential plane images, whereas alternate bright and dark layers were observed along the radial direction and were recognized as elastic laminas (ELs) and smooth muscle-rich layers (SMLs), respectively. Elastin and collagen fibers are mainly oriented in the circumferential direction, and waviness of collagen fibers was significantly higher than that of elastin fibers. Collagen fibers were more undulated in longitudinal than in radial direction, whereas undulation of elastin fibers was equibiaxial. Changes in waviness of collagen fibers during pressurization were then evaluated using 2-dimensional fast Fourier transform in mouse aortas, and indices of waviness of collagen fibers decreased with increases in intraluminal pressure. These indices also showed that collagen fibers in SMLs became straight at lower intraluminal pressures than those in EL, indicating that SMLs stretched more than ELs. These results indicate that deformation of the aorta due to pressurization is complicated because of the heterogeneity of tissue layers and differences in elastic properties of ELs, SMLs, and surrounding collagen and elastin.     

The infrastructure of aortic elastic fibers

Elastic fibers are composed of a central core of elastin that is amorphous and electron-lucent in conventional transmission electron micrographs and peripheral microfibrils. A complex infrastructure within the amorphous elastin of mature rat aorta is made visible by fixation and staining with a glutaraldehyde-ruthenium red mixture in phosphate buffer or osmium-ruthenium red in cacodylate buffer. The infrastructure is composed of at least two interlacing but distinct elastic structural components; a framework of circumferentially orientated microfibrils and a three-dimensional meshwork of filaments that permeate the fiber. The latter resembles a reticulum that has previously been observed in freeze-fractured and negatively stained elastin and attributed to the supramolecular organization of elastin. Microfibrils also extend from the core of the elastic fiber into the surrounding matrix where they appear to function as anchoring fibers. These observations indicate that the elastic properties of the arterial wall are an integrated function of both elastin and microfibrils.     

Pattern of accumulation of elastin and the level of mRNA for elastin in aortic tissue of growing chickens

Synthesis and accumulation of elastin in many elastic tissues begins in the last third of fetal development, reaches a maximum shortly after birth, and then declines rapidly. For the aorta of the chick and the pig and the ligamentum nuchae and lung of the sheep, it has been shown that increased levels of elastin production with fetal development are correlated with increased levels of elastin mRNA in the tissue, measured both by cell-free translation and by hybridization to cDNA probes. In this study we examine the relationship between insoluble elastin accumulation and message levels for tropoelastin in aortic tissue of chickens during posthatching development and growth. Whether evaluated by cell-free translation or by dot blot hybridization, steady state levels of tropoelastin message increase to a maximum at 2 weeks after hatching, and then fall rapidly with further development and growth. This pattern correlates well with production of insoluble elastin by the aorta, determined either by direct measurements of synthesis or by rate of accumulation of insoluble elastin. The data indicate that the major site of regulation of elastin production is pretranslational throughout the entire period of development and growth of the chicken aorta.     

Semicarbazide-sensitive amine oxidase in annulo-aortic ectasia disease: relation to elastic lamellae-associated proteins.

Lysyl oxidases (Lox), which are members of the amine oxidase family, are involved in the maturation of elastic lamellae and collagen fibers. Modifications of amine oxidases in idiopathic annulo-aortic ectasia disease (IAAED) have never been investigated. Our aim was to examine the expression of several proteins that might interfere with elastic fiber organization in control (n=10) and IAAED (n=18) aortic tissues obtained at surgery. Expression of amine oxidases and semicarbazide-sensitive amine oxidase (SSAO), and cellular phenotypic markers were examined by immunohistopathology and confocal microscopy. The expression of these proteins was assessed in relation to clinical and histomorphological features of the arterial wall. In control aorta, SSAO staining was expressed along elastic lamellae, whereas in aneurysmal areas of IAAED, SSAO was markedly decreased, in association with severe disorganization of elastic lamellae. Smooth muscle myosin heavy chain was also decreased in IAAED compared with controls, indicating smooth muscle cell dedifferentiation. Multiple regression analysis showed that elastic lamellar thickness (ELT) was correlated positively with the SSAO:elastin ratio and negatively with the Lox:elastin ratio, and that the clinical features of IAAED (aneurysm, thoracic aorta diameter, and aortic insufficiency) were positively correlated with ELT but not with SSAO. The relationship between SSAO expression and ELT suggests that this amine oxidase may be involved in elastic fiber organization. However, in advanced IAAED, the deficit in SSAO expression could be secondary to the decrease and fragmentation of elastic fibers and/or to vascular smooth muscle cell dedifferentiation.     

Structural strain energy function applied to the ageing of the human aorta

Stiffening of the aorta with progressing age leads to decrease of aortic compliance and thus to an increase of pulse pressure amplitude. Using a strain energy function (SEF) which takes into account the composition of the arterial wall, we have studied the evolution of key structural components of the human thoracic aorta using data obtained from the literature. The SEF takes into account the wavy nature of collagen, which upon gradual inflation of the blood vessel is assumed to straighten out and become engaged in bearing load. The engagement of the individual fibers is assumed to be distributed log-logistically. The use of a SEF enables the consideration of axial stretch (lambda(z)) and residual strain (opening angle) in the biomechanical analysis. Both lambda(z) and opening angle are known to change with age. Results obtained from applying the SEF to the measurements of aortic pressure-diameter curves indicate that the changes in aortic biomechanics with progressing age are not to be sought in the elastic constants of elastin and collagen or their volume fractions of the aortic wall but moreover in alterations of the collagen mesh arrangement and the waviness of the collagen fibers. In old subjects, the collagen fiber ensemble engages in load bearing much more abruptly than in young subjects. Reasons for this change in collagen fiber dynamics may include fiber waviness remodeling or cross-linkage by advanced glycation end-products (AGE). The abruptness of collagen fiber engagement is also the model parameter that is most responsible for the decreased compliance at progressed ages.     

Synchrotron radiation X-ray fluorescence microscopy reveals a spatial association of copper on elastic laminae in rat aortic media

Copper, an essential trace metal in humans, plays an important role in elastic formation. However, little is known about the spatial association between copper, elastin, and elastin producing cells. The aorta is the largest artery; the aortic media is primarily composed of the elastic lamellae and vascular smooth muscle cells, which makes it a good model to address this issue. Synchrotron radiation X-ray fluorescence microscopy (SRXRF) is a new generation technique to investigate the spatial topography of trace metals in biological samples. Recently, we utilized this technique to determine the topography of copper as well as other trace elements in aortic media of Sprague Dawley rats. A standard rat diet was used to feed Sprague Dawley rats, which contains the normal dietary requirements of copper and zinc. Paraffin embedded segments (4 μm of thickness) of thoracic aorta were analyzed using a 10 keV incident monochromatic X-ray beam focusing on a spot size of 0.3 μm × 0.2 μm (horizontal × vertical). The X-ray spectrum was measured using an energy-dispersive silicon drift detector for elemental topography. Our results showed that phosphorus, sulfur, and zinc are predominately distributed in the vascular smooth muscle cells, whereas copper is dramatically accumulated in elastic laminae, indicating a preferential spatial association of copper on elastic laminae in aortic media. This finding sheds new light on the role of copper in elastic formation. Our studies also demonstrate that SRXRF allows for the visualization of trace elements in tissues and cells of rodent aorta with high spatial resolution and provides an opportunity to study the role of trace elements in vasculature.     

The effect of developing hypertension on the synthesis and accumulation of elastin in the aorta of the rat

This report describes an investigation of the effects of developing hypertension on the synthesis and accumulation of insoluble elastin in the thoracic aorta of young rats. Uninephrectomized male rats were made hypertensive by administration of deoxycorticosterone acetate and addition of 1% NaCl to their drinking water. Divergence of systolic blood pressures between treated and control animals and hypertrophy of the vessel began after about 2 weeks of treatment. Coincident with the appearance of hypertrophy, there was an increased accumulation of insoluble elastin in the aorta and a large increase in the capacity of the aortic tissue to synthesize elastin. However, in spite of continued increases in blood pressure and vessel hypertrophy, this effect on elastin synthesis and accumulation was transient. The results of this study suggest that synthesis of elastin in aortic tissue of young rats is highly sensitive to alterations in blood pressure.     

Electron microscopic study of the prenatal development of the thoracic aorta in the rat

Prenatal development of the thoracic aorta of the rat during the period ranging from gestational days 12 to 21 was examined by transmission electron microscopic and morphometric studies. The process of wall formation occurred in four major phases. At phase I (gestational day 12), the dorsal aorta consists of an endothelium and loosely surrounding mesenchymal cells. Collagen fibrils and fine filamentous materials are sparsely present in the intercellular space. At phase II (days 13 to 16), the mesenchymal cells begin to differentiate to myoblasts, which have small clusters of myofilaments with dense bodies, rough endoplasmic reticulum, and a discontinuous basal lamina. The differentiating cells form a few compact cell layers around the endothelium. Elastic fibers first occur sparsely in juxtacellular spaces at days 13-14. The thickness of the aorta increases rapidly from 1-3 layers of cells at day 13 to 5-8 layers at day 17, leading to a maximum of 5-9 cell layers at day 20. The differentiation of myoblasts and elastogenesis are initiated in the inner layers, and later progress toward the outer layer of the aortic wall. At phase III (days 17 to 19), the myoblasts continue to develop into typical smooth muscle cells, and elastic fibers rapidly increase in both size and number. At phase IV (day 20 and later), smooth muscle cells have well-developed myofilaments in the cell periphery, and rough endoplasmic reticulum and other organelles tend to accumulate in the apical portion of the cytoplasm. Elastic laminae appear in a few inner layers of the aortic wall.     

Chronic administration of minoxidil protects elastic fibers and stimulates their neosynthesis with improvement of the aorta mechanics in mice

Arterial wall elastic fibers, made of 90% elastin, are arranged into elastic lamellae which are responsible for the resilience and elastic properties of the large arteries (aorta and its proximal branches). Elastin is synthesized only in early life and adolescence mainly by the vascular smooth muscles cells (VSMC) through the cross-linking of its soluble precursor, tropoelastin. In normal aging, the elastic fibers become fragmented and the mechanical load is transferred to collagen fibers, which are 100–1000 times stiffer than elastic fibers. Minoxidil, an ATP-dependent K⁺ channel opener, has been shown to stimulate elastin expression in vitro, and in vivo in the aorta of male aged mice and young adult hypertensive rats. Here, we have studied the effect of a 3-month chronic oral treatment with minoxidil (120 mg/L in drinking water) on the abdominal aorta structure and function in adult (6-month-old) and aged (24-month-old) male and female mice. Our results show that minoxidil treatment preserves elastic lamellae integrity at both ages, which is accompanied by the formation of newly synthesized elastic fibers in aged mice. This leads to a generally decreased pulse pressure and a significant improvement of the arterial biomechanical properties in female mice, which present an increased distensibility and a decreased rigidity of the aorta. Our studies show that minoxidil treatment reversed some of the major adverse effects of arterial aging in mice and could be an interesting anti-arterial aging agent, also potentially usable for female-targeted therapies.     

Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts

The formation of a mature elastic fiber is thought to proceed by the deposition of elastin on pre-existing microfibrils (10-12 nm in diameter). Immunohistochemical evidence has suggested that in developing tissues such as aorta and ligamentum nuchae, small amounts of elastin are associated with microfibrils but are not detected at the light microscopic and ultrastructural levels. Dermal tissue contains a complex elastic fiber system consisting of three types of fibers--oxytalan, elaunin, and elastic--which are believed to differ in their relative contents of microfibrils and elastin. According to ultrastructural analysis, oxytalan fibers contain only microfibrils, elaunin fibers contain small quantities of amorphous elastin, and elastic fibers are predominantly elastin. Using indirect immunofluorescence techniques, we demonstrate in this study that nonamorphous elastin is associated with the oxytalan fibers. Frozen sections of normal skin were incubated with antibodies directed against human aortic alpha elastin and against microfibrillar proteins isolated from cultured calf aortic smooth muscle cells. The antibodies to the microfibrillar proteins and elastin reacted strongly with the oxytalan fibers of the upper dermis. Oxytalan fibers therefore are composed of both microfibrils and small amounts of elastin. Elastin was demonstrated extracellularly in human skin fibroblasts in vitro by indirect immunofluorescence. The extracellular association of nonamorphous elastin and microfibrils on similar fibrils was visualized by immunoelectron microscopy. Treatment of these cultures with sodium dodecyl sulfate/mercaptoethanol (SDS/ME) solubilized tropoelastin and other proteins that reacted with the antibodies to the microfibrillar proteins. It was concluded that the association of the microfibrils with nonamorphous elastin in intact dermis and cultured human skin fibroblasts may represent the initial step in elastogenesis.     

Ultrastructural localization of Helix pomatia lectin-binding sites in mouse lung elastic fibers

Helix pomatia (Snail) lectin complexed with colloidal gold (HPL-gold) recognized binding sites on elastic fibers in plastic embedded sections of lung tissue from mice of several ages. Deposition of the lectin-gold particles was examined by electron microscopy. Structures such as the elastic laminae of pulmonary vessels and elastic fibers throughout the lung was specifically and intensely decorated by the HPL-gold complex and easily visualized. The binding of the HPL-gold particles was primarily to sites on the amorphous component of elastin, to the virtual exclusion of the microfibrillar elastin elements, collagen fibers and other components of the extracellular matrix. In addition, moderate age differences in the binding of HPL-gold to elastin were apparent. These observations appear to be the first demonstration of the presence, in the amorphous component of elastin, of glycoconjugates that are specifically recognized by HPL and suggest a method by which the involvement of glycoconjugates in lung elastogenesis could be explored.     

Elastic laminae in vascular development and disease

The activities of vascular cells, including adhesion, proliferation, and migration, are mediated by extracellular matrix components, including collagen matrix and elastic fibers or laminae. Whereas the collagen matrix stimulates vascular cell adhesion, proliferation, and migration, the elastic laminae inhibit these activities. Coordinated regulation of cell activities by these matrix components is an essential process for controlling the development and remodeling of the vascular system. This article summarizes recent development on the role of arterial elastic laminae in regulating the development of smooth muscle-like cells from bone marrow-derived progenitor cells as well as in mediating cell adhesion, proliferation, and migration with a focus on the molecular mechanisms and physiological significance.